The long-term goal of this project is the characterization of neurotransmitter receptor-mediated information transduction, and its regulation, across neuronal membranes. The primary, but not exclusive, model systems under investigation are those for dopamine receptors. In order to characterize dopamine and related receptors at the biochemical and molecular levels and study their regulation, there are two major interrelated lines of research which are ongoing: 1) investigation of the cell biology, function and regulation of the receptors at the protein level; and 2) molecular cloning of the receptor cDNAs/genes and investigation of receptor structure and regulation in normal and pathophysiological states. 1) Cell Biology and Regulation of Dopamine Receptors: Characterization of the functional and regulatory properties of D-1 and D-2 dopamine receptors in various cDNA-transfected cell lines was continued. The D-1A and D-1B receptors were shown to undergo agonist-induced desensitization in CHO cells. The role of cAMP in this response is being tested using mutagenesis techniques. Both short and long isoforms of the D-2 receptor were also shown to undergo agonist-induced desensitization in CHO cells. The D-2S receptor was also down-regulated by agonist treatment whereas the D-2L receptor was up-regulated through a mechanism involving increased receptor synthesis. Both D-2 receptor isoforms were expressed in NG108-15 neuroblastoma cells and shown to couple to K+ channels, albeit through different G proteins. The D-1A and D-3 receptor proteins were mapped in the CNS using immunohistochemical methods. 2) Molecular Cloning of Dopamine and other Receptors: Both D-1 receptor subtypes were sequenced in the spontaneous hypertensive rat (5HR) which exhibits defective kidney D-1 receptors. No differences in sequence were found in comparison to control rats. Work continued on the cloning of a third "D-1 like" receptor which apparently is linked to the stimulation of phosphatidylinositol turnover and mobilization of calcium. Transgenic "knock-out" mice lacking a functional D-1A receptor were produced and are undergoing characterization. Other transgenic mice lacking the D-1B and D-3 receptors are in production. Chimeric D- 2/D-3 and D-2/D-4 dopamine receptors were constructed and expressed for characterization. The 5-HT-6 and 5-HT-7 serotonin receptor subtypes were further characterized with respect to their pharmacology and regulatory properties. A cDNA clone encoding an opiate-like receptor was expressed and characterized.